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LSU Historical Dissertations and Theses
Graduate School
1968
Chemical, Physical and Biological Properties of
Four Strains of Sugarcanemosaic Virus.
William Payton Bond
Louisiana State University and Agricultural & Mechanical College
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Bond, William Payton, "Chemical, Physical and Biological Properties of Four Strains of Sugarcanemosaic Virus." (1968). LSU
Historical Dissertations and Theses. 1434.
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6 8-16,302
BOND, William Payton, 1941CHEMICAL, PHYSICAL AND BIOLOGICAL PROPERTIES
OF FOUR STRAINS OF SUGARCANE MOSAIC VIRUS.
Louisiana State University and Agricultural and
Mechanical College, Ph.D„ 1968
Agriculture, plant pathology
University Microfilms, Inc., Ann Arbor, Michigan
CHEMICAL, PHYSICAL AND BIOLOGICAL PROPERTIES
OF FOUR STRAINS OF SUGARCANE MOSAIC VIRUS
A Dissertation
Submitted to the Graduate Faculty of the
Louisiana State University and
Agricultural and Mechanical College
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
in
• The Department of Botany and Plant Pathology
by
William Payton Bond
B.S., Southeastern Louisiana College, 1963
M.S., Louisiana State University, 1966
May, 1968
ACKNOWLEDGMENT
The writer wishes to express his sincere appreciation to Dr.
Thomas P. Pirone, under whose guidance this research has been con­
ducted.
The writer also wishes to express his sincere appreciation
to Dr. I. L. Forbes for his constructive criticism in the prepara­
tion of this manuscript and for serving as committee chairman in the
absence of Dr. Pirone.
Appreciation is extended to Dr. M. T.
Henderson, Dr. Gordon Holcomb,
and Dr. Louis Anzalone, members of
the examining committee.
Appreciation is also extended to Dr. S. J. P. Chilton, Head,
Department of Botany and Plant Pathology for making funds and
facilities available.
The writer wishes to thank John Ivey for the photographic work. .
ii
TABLE OF CONTENTS
Page
ACKNOWLEDGMENT..........................
ii
LIST OF TABLES............... •.........................
v
vi
LIST OF PLATES...............................
LIST OF F I G U R E S ...................................................
A BSTRACT........................................
INTRODUCTION.
viii
. . .................................................
LITERATURE REVIEW .................................
.
Virus Strains and Source P l a n t s .................... ■
Virus Assay..............................................
Differential V a r i e t i e s ................... .............
Symptoms on Sorghum Produced by the Different Strains.
Physical Properties of Strains of S C M V ..........
15
Comparison of Three Methods of Virus Purification. . .
Serology of Strains of SCMV. . .
.....................
Soil Transmission Studies................... ...........
EXPERIMENTAL RESULTS.
I.
II.
III.
IV.
V.
VI.
VII.
I.
II.
III.
.............' ..............
..........
14
14
14
14'
15
16
19
21
24
Reaction of Virus Strains on Standard Differential
Varieties.................................................
Symptoms on Sorghum Produced by the Different Strains.
Physical Properties of Strains of S C M V ................
Comparison of Three Methods of Virus Purification
Usefulness of the Three Procedures for Purification
of Different Strains
.............................
Serology of Strains of S C M V .................. ...
Soil Transmission Studies...............................
DISCUSSION.
1
4
MATERIALS AND METHODS .............................................
I.
' II.
III.
IV.
V.
VI.
VII.
VIII.
vii
24
24
24
33
33
38
. . .
46
Physical Properties of Strains
of S C M V ................
Serology of Strains of S CMV ..............................
Soil Transmission of S C M V .................... ..........
47
50
51
SUMMARY . . . . . .
...............................................
iii
54
Page
LITERATURE C I TED ................................................
.
V I T A .................................................................
iv
56
63
LIST OF TABLES
TABLE
1.
2.
Page
Results of inoculation experiments showing infection
ratio of strains A, B, D, H, and Johnson grass mosaic
in standard differentials.................................
25
Thermal inactivation points of strains A, B, D, and H
of SCMV in sorghum.........................................
27
3. . Dilution end point of strains A, B, D, and H of SCMV
in s o r g h u m ...........
4.
5.
6.
7.
8.
9.
10.
29
Procedures used in purification by different methods
after which assays were made
..............
30
Comparative infectivity of SCMV after treatments listed
in Table 3 .................................................
31
Microprecipitin reactions of sugarcane mosaic virus
(SCMV) strains against antisera to strains A, B, D,
and H ...................................
34
••
Transmission of sugarcane mosaic virus (SCMV) from
infected plants to adjacent noninoculated sorghum
p l a n t s .................................
39
Transmission of sugarcane mosaic virus (SCMV) from
infected plants to noninoculated sorghum plants through
soil water (Method A ) ......................................
41
Transmission of sugarcane mosaic virus (SCMV) from
infected plants to noninoculated plants through soil
water (Method B ) ..........................................
42
Transmission of sugarcane mosaic virus (SCMV) in sorghum
plants under screened cages in the absence of root
contact. . . . . . .
........................... . . . . .
43
v
LIST OF FIGURES
FIGURE
1.
2-5.
6-10.
Page
Absorption spectrum of a purified preparation of
sugarcane mosaic virus.
The curve is a tracing
using a Perkin Elmer Spectrophotometer ....................
32
Agar diffusion tests with strains of SCMV.
2)
Precipitin bands produced between peripheral
wells containing degraded virus protein of strains A,
B, D, H and Johnson grass mosaic and healthy sorghum
protein and the center well containing antiserum
against strain A.
3) Center well containing anti­
serum against strain B. . 4) Center well containing anti­
serum to strain D. 5) Center well containing antiserum
against strain H ..................'.......................
36
Agar diffusion tests with strains of SCMV.
6) Preci­
pitin bands produced between peripheral wells contain­
ing antisera against strains A, B, D, and H and
Johnson grass mosaic and the center well containing
degraded virus protein of strain A.
7) Center well
containing degraded virus protein of strain B.
8) Center well containing degraded virus protein of
strain D.
9) Center well containing degraded virus
protein of strain H.
10) Center well containing
degraded virus protein of strain H ......................
37
vi
LIST OF PLATES
PLATE
1.
2.
Page
Reaction of beefbuilder sorghum to strains A, B, D
and H of sugarcane mosaic virus (SCMV) when grown
under conditions of low temperature and high humidity.
Left to right strains A, B, D, and H .....................
26
Necrosis on roots of surface sterilized sorghum seeds
germinated in sterile w a t e r ......... .....................
45
vii
ABSTRACT
Several strains of sugarcane mosaic virus
described using differential host varieties.
(SCMV) have been
In studies reported
here, an attempt was made to separate the four common strains of
SCMV (A, B, D, and H) using chemical, physical and biological proper­
ties.
.
•Studies of the physical properties of the four strains showed
that thermal inactivation points (TIP) are of no value in strain differentation.
All strains were still active at 55°C, but not at 57°C.
Dilution end point
(DEP) studies revealed a difference in certain of
the virus strains in their tolerance to dilution.
A
were still infectious at 10
Strain A and H
—Q
, strain D at 10
_0
, and strain B at 10
.
A severe leaf necrosis developed on plants infected with cer­
tain of the virus strains.
Necrosis occurred on sdrghum pT'ants .‘
i nfected
with strains A, D and H, but not on plants infected with strain B.
There was a correlation between the presence of the leaf necrosis
and virus concentration.
Three methods of purification were compared to determine the
one best suited for use with SCMV.
A modification of the method of
Delgado-Sanches and Grogan for potato virus Y yielded the highest
amount of infectious virus.
Virus purified by this method had less
host contaminating material than with other methods tested.
Serological studies were made of the four SCMV strains as' well
as the Johnson grass mosaic.
Results obtained from.microprecipitin
viii
tests showed that none of the virus strains could be differentiated
using this technique.
closely related.
In these tests, all strains appeared to be
Agar diffusion tests showed that strains A, D and
H are closely related.
Antigen of strain B did not react with
antisera to any of the virus strains including its own.
Micropre­
cipitin and agar diffusion tests showed that the Johnson grass mosaic
in Louisiana is serologically related to SCMV, but not as closely as •
are the strains to each other.
Spurring in agar diffusion tests
indicate that it is a distinct strain of SCMV.
Studies showed that SCMV can be transmitted from infected plants
to noninoculated plants through the soil.
the absence of root contact.
Transmission occurred in
The involvement of a biological vector
in soil transmission remains to be demonstrated.
ix
INTRODUCTION
Sugarcane mosaic virus
(SCMV), affects sugarcane (Saccharum
officinarum L.) and certain other members of the Gramineae.
The
disease caused by this virus has been known for over 70 years,
although,
Brandes
it was not until 1920 that its viral nature was shown by
(17).
Electron microscopy of leaf dip and partially purified prepara-tions have shown SCMV to be a flexuous rod with a length of about
750 m u (32, 38, 57).
This places SCMV in the potato virus y (PVY)
group of plant viruses in Brandes1 (18) system of classification.
The viruses in this group are all serologically related and have
"normal lengths" of 730-790 mu.
SCMV,
strains.
like most viruses, is probably composed of a number of
Several strains of SCMV have been described through the use
of differential host varieties.
and Rands
Summers
(72, 73) and Summers, Brandes
(74) described ten strains and substrains based on the
symptoms produced on the sugarcane varieties Co. 281, C.P. 29-291
and C.P. 31-294.
Abbott and Tippett (5) used C.P. 31-294 to differen­
tiate strains A, 3, D, E, and F, and C.P. 31-588 to differentiate
strains A and H.
The use of differential host varieties has been used effectively
to differentiate strains of certain plant viruses.
However,
several
workers are of the opinion that differentiation of strains on the
basis of macroscopic symptoms is of limited usefulness
(5, 13).
Bawden (12) states that "rnany virus workers are reluctant to appre­
ciate' that variability, especially (in symptomatology and host range,
is normal rather than exceptional."
Factors such as environmental
conditions affect the symptoms produced and unless the environment
is defined, conditions may be described which may never again be pr e ­
cisely reproduced.
According to Bennett
(13), serology is more
accurate than differential host varieties in establishing strain
relationships.
Serology has been used by workers in differentiating strains of
many plant viruses
(11, 31, 34, 65, 86).
Bawden (12) is of the opinion
that serology is the most useful and accurate means of allocating
strains to a given collective species.
The use of serology was
limited by early failures to demonstrate serological reactions with
some viruses.
Recently, better techniques for preparing antigens,
along with better serological tests have made serology more applicable
for use with plant v i r u s e s . '
Desai (27), Perez and Adsuar (56) were the first to show the
antigenic nature of SCMV.
Perez and Adsuar
(56) and Abbott and
Tippett (5) suggested that serology might be used in demonstrating
possible strain relationships.
Until recently, however,
serological
studies with SCMV have not been feasible due to the absence of a suit­
able method of virus purification.
Recently,
Pirone and Anzalone (57)
and Shepherd (66) have purified SCMV and have obtained antisera of
sufficiently high titer for use in serological studies.
The physical properties of a virus are often helpful in differ-,
entiation of strains.
A number of workers have investigated the
physical properties of SCMV (1, 5, 6, 22, 23) and have reported a.
wide range of values, both for the thermal inactivation point (TIP)
and the dilution end point (DEP).
Abbott and Tippett(5) in a recent
study of the physical properties of SCMV
ences in
strains, reported differ­
the TIP and DEP values for different strains, but concluded
that the use of physical properties in strain differentiation was
limited.
This paper describes a study of the chemical, physical and
biological properties of four common strains of SCMV.
Also, the
Johnson grass strain (68, 83) of SCMV, recently reported in Louisiana
(58) was included to determine its relationship to the SCMV.
During the course of this study, certain events indicated that
SCMV was
being transmitted from plant to plant through the soil.
Tests were made to determine if
borne manner.
SCMV was being transmitted in a soil-
LITERATURE R E V I E W
According to Matz (49), mosaic was first reported as a sugar­
cane abnormality by V o n Musschenbroek in Java in 1892 where it was
known as "gel'estrepenzike" or yellow stripe disease.
Although the
disease had been k n own for a long time, it was not until 1920 that
/
its infectious nature was demonstrated.,-
Brandes
(17) was able to
transmit the disease both mechanically and with insects.
showed for the first time, under controlled conditions,
Brandes
that the cell
sap of diseased plants is infectious when introduced into the young
tissues of healthy plants.
The symptoms of mosaic, which vary in intensity on different
varieties, are usually an irregular mottling, with islands of darker
green on a background of paler green or yellowish chlorotic areas
(28).
Edgerton (28) states that symptoms are influenced by the cane
variety,
the condition of growth, the temperature, and the strain of
the virus involved.
•
The host range of SCMV is limited to members of the grass family.
Summers, Brandes, and Rands
hosts of SCMV.
(74) list 10 cultivated and 34 wild grass
Four of the records on cultivated hosts represented
observations and six were experimental transmissions, while 19 of
the records on wild hosts were observations and 16 were experimental
transmissions.
The cultivated grasses reported as. susceptible to
SCMV by experimental transmission are:
4
Andropogon sp., Miscanthus
sinensis Anderss, Pennisetum glaucum (L.) R. Br., Sorghum vulgare var.
sudanense
(Piper) Hitch., and Zea mays L.
susceptible experimentally are:
The wild grasses reported
Digitaria sanguinalis (L.) Scop.,
Digitaria violascens (L.) Link, Echinocloa colonum (L.) Link,
Echinocloa crusgalli (L.) Beauv., Eleusine indica
(L.) Gaertn.,
Erianthus giganteus (Walt.) Muhl, Lamarckia aurea (L.) Moench, Narenga
porphyrocoma
(Hance) Bor., Panicum dichotomiflorum Michx.. Paspalum
boslanum Flugge, Paspalum fimbriaturn H. B. K . , Paspalum virgatum L.,
Setaria lutescens
poiretiana
Anzalone
(Weigal) F. T. Hubb,
Setaria magna Griseb,
(Schult.) Kunth, and Setaria verticulatta
(L.) Beauv.
(7) in 1963 found four cultivated varieties of rice
sativa L.) susceptible to strain H of SCMV.
Setaria
(Oryza
Todd (79) in 1964
reported St. Augustine grass (Stenotaphrum secundatum (Waltz.),
Kuntze) susceptible to SCMV.
Also,
in 1964 Abbott and Tippett (4)
using four strains of SCMV, found Andropogoh virginicus L., Sorghum
halepense (L.) Pers., Triticum aestivum L., Secale cerale L . , and
Hordeum vulgare L. susceptible to SCMV.
(55) found Raoul grass
Recently,
Perdomo and Forbes
(Rottboellia exaltata L. F.) susceptible to
SCMV.
SCMV is transmitted mechanically and by several aphid species
(74).
Brandes
The_virus has a stylet-borne relationship with aphid vectors.
(17) in 1920 demonstrated that Rhopalosiphum maidis Fitch was
able to transmit the virus to healthy plants after a feeding period on
infected plants.
R. maidis was the only known vector of SCMV until
1933 when Ingram and Summers (41), in preliminary experiments,
showed
that the rusty plum aphid, Hysteroneura setarie Thos. was also capable
of transmitting the virus.
Ingram and Summers
(42) in 1938 reported
that Toxoptera graminum Rond..could also transmit the virus.
Tate
and Vandenburg (75) in 1939 reported Carolinaia cyperi Ainslie as a
vector in Puerto Rico.
Recently,-other aphids have been shown to
transmit SCMV.
They are:
ambrosiae Thos.
(3), Amphorophora sonchi Destl.
Sulz.
Acyrthosiphon pisum Harr.
(3), Dactynotus
(3), and Myzus persicae
(8).
Partially purified preparations from mosaic infected sugarcane
and corn yielded rod-shaped virus particles which had an average
diameter of 15 mu and a length of 620-670 mu (57, 66).
Weibel
Herold and
(38) reported rod-shaped virus particles averaging 760 £ 10 mu
in length and 12-13 mu in diameter from leaf dip preparations.
and Anzalone
Pirone
(57), using the concept of normal length as set forth by
Brandes and Wetter
(19), found that partially purified preparations
from juice of mosaic infected sorghum yielded long flexuous rod-shaped
virus particles with a normal length of 755 mu.
The fact that SCMV
particles have normal lengths of approximately 750 mu places the virus
in the potato virus Y (PVY) group in Brandes'
(18) system of classi­
fication.
Several strains of SCMV have been described (2, 74).
These
strains were identified according to their reaction on certain dif­
ferential varieties
(5, 74).
Tims and Edgerton (77) were the first
to mention the possibility of strains of the virus. • This hypothesis •
was based on the differences in degree of infection with mosaic,
observed in four varieties of sugarcane at two localities in
Louisiana.
Storey (71) in 1927 reported that he was able to separate
two supposed strains of SCMV.
The identification.of these strains
was based upon differences in regional distribution and host range
in Natal,
South Africa.
Tims, Mills,
and Edgerton (78) in 1935
reported differences in virulence between the viruses from areas of
heavy and light mosaic incidence.
They concluded that "two very dis­
tinct types of mosaic, recognized by very distinct symptoms, occur in
Louisiana.11
Summers
(72).also in 1935 described four strains which
were designated 1, 2, 3, and 4.
These strains were differentiated
principally by symptoms produced on the sugarcane variety C.P. 28-60.
He later reported (73) seven strains which were designated as A, B,
C, D, E, F, and G, and three substrains of D, with strains A, B, C,
and D corresponding to the previously designated 1, 2, 3, and 4.
The differentiation of these strains was based on symptoms produced
on the sugarcane varieties, C.P. 31-294, C. P. 29-291, and Co. 281.
Summers, Brandes and Rands (74) in 1948 explained in detail the
experiments that led to the differentiation of the 10 strains and sub
strains and furnished a key for their identification on Summers'
differential host varieties.
Liu (46) in 1950 described four strains of SCMV in Taiwan,
designating them A, B, C, and D.
Since the differential hosts used
were different from those used by Summers,
no comparison can be made
between these strains and those described by Summers.
Later, Liu
and Li (47) reported the existence of only three strains of SCMV in
Taiwan.
These were designated as "short stripe type
stripe type (YS)," and "fine-stripe type (FS)."
(SS)," "yellow-
Abbott (2) in 1961 reported a new strain of SCMV designated as
"strain H.
This strain is considered to be the most severe strain due
to its ability to attack several varieties of sugarcane previously
considered immune to mosaic.
Abbott and Tippett (5) in 1966 reported the results of a study
of Summers'
stock cultures of five strains and four substrains on
various differential hosts.
These authors reported that strains A,
B, D, and H could be differentiated using the differential host varie­
ties C.P. 31-588 and C.P. 31-294.
In this study, Co. 281 was
excluded because it differentiates only strain C which is rare.
According to these workers,
this strain can be identified in the
field without transfer to differential hosts.
In 1963, a new mechanically transmissible virus was isolated
from corn in Ohio (84).
Since then, a similar virus disease has been
reported from a number of states (24, 58, 69).
has been called maize dwarf mosaic vitus
(85) because of the dwarfing
symptoms which it supposedly produces on corn.
(85) reported that
virus,
MDMV
This virus disease
Williams and Alexander
had properties similar to sugarcane mosaic
although, no relationship was shown in their preliminary
serological tests.
Recently,
Shepherd (66) and Bancroft et
al.
(11)
have shown that MDMV and SCMV are morphologically similar and sero­
logically related.
Wagner and Dale (83) tested several isolates of
MDMV from several states and found that all were serologically related
to SCMV.
These authors have suggested that MDMV is probably a strain
of SCMV.
This virus, however is unique in that it readily infects
Johnson grass
(Sorghum halepense)
(84).
For this reason, it is
sometimes referred to as the Johnson grass strain of SCMV (68).
Sugarcane is highly refractory to infection by this strain (25).
A number of workers have investigated the physical properties
of SCMV (1, 5, 6, 22, 23, 45).
inconsistent.
Chona
However,
(22) in 1944 reported thermal inactivation
points of 45, 55, and 65°C, respectively,
which he worked.
of 55°C.
the data presented is very
for the three strains with
Adsuar (6) reported a thermal inactivation point
Costa and Penteado (23) also reported a thermal inactiva­
tion point of 55°C.
Abbott (1) in 1953 reported that all strains of
SCMV were inactivated at 53°C.
Recently, Abbott and Tippett (5)
reinvestigated the physical properties to determine if physical
properties might supplement macroscopic symptoms in strain differ- entiation.
In this study,
thermal inactivation points were determined
for three strains and eight variants of SCMV.
The thermal inactiva­
tion points for strains A, D, and H were 53, 52, and 49°C, respectively.
However,
the fact that inactivation was obtained at one
temperature and regained at a higher temperature might lead one to
question the validity of these results.
A wide range of values has also been reported for the dilution
end point
(DEP) of SCMV (1, 5, 23, 45, 63).
Rafay (63) in 1935
reported a dilution end point of 10"^ for SCMV.
Lawas and Fernandez
(45) in 1949 also reported 10"! as the dilution end point.
Penteado
Costa and
(23) reported a value of 10“-* for juice extracted from corn
infected with SCMV.
In 1953, Abbott (1) reported a dilution end
„3
point of 10
for six strains which he studied.
Recently, Abbott
and Tippett (5) made dilution end point studies of strains A, D,
*
10
and H.
They reported values of 10“ 3 for strains A and D and 10"^ for
strain H.
Desai (27) and Perez and Adsuar (56) were the first to s h o w that SCMV is antigenic.
Perez and Adsuar suggested the possibility
of using the precipitin reaction in testing for relationships among
strains of SCMV.
Recently, Abbott "and Tippett
(5) concluded that
differentiation of strains of SCMV on the basis of symptoms and
physical properties was limited, and suggested that serology might
be of value in strain differentiation.
Until recently, however,
this
has not been feasible due to the absence of a suitable method of puri­
fication.
Pirone and Anzalone
(57) and Shepherd (66) have purified
SCMV, and have obtained antisera of sufficiently high titer for use
in serological studies.
Serology has proven useful in establishing relationships of
many plant viruses (11, 31, 34, 65, 86).
Bennett (13) in 1953
reported that serology was more accurate than differential hosts and
physical properties in differentiating strains of most plant viruses.
According to Ball (10), Dvorak was the first to apply serological
methods to plant viruses.
Purdy (62) in 1928, using precipitin and
complement fixation tests, showed the specificity of serological reac­
tions.
Birkeland (15) was the first to show that strains of plant
viruses contained specific antigens which differentiated them from
other members of the group.
Chester
(21) in 1936 used the cross
absorption technique to demonstrate serological differences among
strains of the same virus.
Despite these findings,
the difficulty
in obtaining antisera of high titer, and the large amounts of antigen
11
required for available serological techniques limited the use of
serology in studies of virus relationships.
In r e c e n t ’years,
the
development of new serological techniques has made serology a more
useful tool for determining virus relationships (29, 54, 82).
Microprecipitin tests have been widely used in serological studies
of plant viruses.
Microprecipitin tests require only small amounts of
antigen and antisera (9)..
Scott et al.
(65), using the microprecipitin
test, found that bean pod mottle and red node viruses were not related.
Shepherd (66), and Bancroft et al.
(11) found that MDMV was sero­
logically related to SCMV using the microprecipitin test.
The Ouchterlony agar double diffusion test represents one of the
newest serological techniques.
Van Slogteren (82) was the first to
apply this procedure to plant viruses.
Scott et al.
•
(65) showed that
bean pod mottle and red node viruses were serologically unrelated,
each giving distinct lines of precipitation.
Willison et al.
(86)
used agar double diffusion tests to show the relationship of certain
stone fruit viruses.
Grogan and Kimble
(34) using this procedure
were able to show that severe bean mosaic virus
southern bean mosaic virus
(SvBMV) from Mexico,
(SBMV) and its related strain in cowpea
were serologically related, but not identical.
Recently, Fulton (31)
used agar double diffusion tests to identify and show relationships
of certain stone fruit viruses.
The Ouchterlony double diffusion tests are well adapted for use
with the spherical and shorter rod-shaped plant vituses, however,
long flexuous rod-shaped plant viruses do not diffuse well in agar
gels.
Purcifull and Shepherd (61) have shown that the flexuous
the
12
rod-shaped viruses could be degraded with alkaline buffers into
antigenically active fragments suitable for use in agar gels.
Biological properties are useful in identifying and grouping
viruses.
Such things as host range, manner of transmission (mechan­
ical, insects, and by nematodes and soil fungi) are useful in estab­
lishing relationships.
There are several plant viruses which are known to be soil
transmissible.
Harrison (36) defined soil-borne viruses as those
"with an underground method of natural spread which does not depend
simply on contact between tissues of infected and healthy plants."
According to Harrison (37), Beijerinck in 1898 showed that tobacco
seedlings became infected with tobacco mosaic virus
(TMV) w h e n grown
in soil in which diseased tobacco plants had been grown.
McKinney
However,
(50) is credited with establishing the fact that some viruses
infect plants naturally through their roots.
McKinney (52) speculated
that wheat mosaic virus (WMV) might be transmitted by nematodes,
borne insects,
soil-
fungi, or without a vector from soil or organic particles.
Other virus diseases which were shown to be transmitted by growing
plants in infested soil were;
tobacco rattle virus
(37), grapevine
fan-leaf (37), lettuce big-vein (43), and tobacco necrosis virus
However,
(70).
not much attention was given to soil-borne viruses until
1958, when Hewitt et al.
(39) showed that grapevine fan-leaf virus was
transmitted by the plant parasitic nematode, Xiphinema ind e x .
in 1958 Fry (30), and Grogan et al.
Also
(33) associated Olpidium brassicae,
a fungus, with the transmission of lettuce big v ein virus.
Olpidium was associated with tobacco stunt virus
In 1960,
(40), and with
13
tobacco necrosis virus
(76).
Recently, more convincing evidence has
been presented concerning the transmission of these viruses by
Olpidium (35).
Brakke and Estes
(16) have recently shown a correla­
tion between the presence of Poloymyxa graminis and the transmission
of soil-borne wheat mosaic virus.
(53) and oat mosaic virus
(OMV)
Barley yellow mosaic virus (BYMV)
(20) are also thought to be trans­
mitted in a manner similar to soil-borne wheat mosaic
Some viruses have
of a
been reported to be soil-borne in the absence
biological .vector.Such viruses are;
(48), tobacco mosaic virus
(14).
(35).
tomato
bushy stunt virus
(35), and chlorotic streak of sugarcane
Several workers have advanced the hypothesis that s o i l •trans-
mission could occur through wounds produced by roots growing through
soil
or sand.
Miyamoto (53) proposed that W M V and BYMV could survive
on soil particles and infect cereal hosts without a vector.
Grogan and Campbell
However,
(35) state that "it is practically impossible to
maintain freedom from a fungus, such as Olpidium, under ordinary
greenhouse conditions."
Thus, Grogan and Campbell are of the opinion
that accidental contamination could account for #the reported trans­
missions in the absence of a biological vector.
MATERIALS AND METHODS
I.
Virus Strains and Source Plants
Strains A, B, D, and H of SCMV used in this study were obtained
from the collection maintained at the U. S. Sugarcane Field Station,
Houma, La.
Beefbuilder T sorghum (Sorghum vulgare x Sorghum vulgare
v a r * sudanenis) was used as the source of the virus.
Mechanical
inoculations were made with freshly expressed sap by means of a
gauze pad onto leaves that had been dusted with 600 mesh carborundum.
Source plants were then placed in the greenhouse.
II.
Virus Assay
Since there are no local lesion hosts known for SCMV,
cent infectivity test was employed for assaying the virus.
otherwise noted, dilutions of 10
for assaying the virus.
-0
,10
- 1 - 2
, 10
and 10
-3
the per
Unless
were used
Each dilution was assayed by mechanically
inoculating 50 sorghum seedlings.
Following inoculation,
the seedlings
were returned to the greenhouse for symptom development.
III.
Differential Varieties
Differential sugarcane varieties used by other workers
(5, 74)
were obtained from the U. S. Sugarcane Field Station, Houma, La.,
and inoculation for comparison with the results of these studies.
Thirty-five eyes each, of the differential varieties C.P. 31-294 and
C.P. 31-588 as well as P.O.J. 234 were planted in the greenhouse.
14
15
SCMV strains A, B, D, H, and the Johnson grass strain were inoculated
at the two-leaf stage into 6 or 7 plants each of the two differential
varieties, as well as into P.O.J. 234.
IV.
Symptoms on Sorghum Produced by the Different Strains
Initial symptoms of strains A, B, D, and H on beefbuilder sorghum
were similar to the symptoms described by Edgerton (28).
after initial symptom expression,
However, so.on
a severe leaf necrosis often deve­
loped on the leaves of infected sorghum plants.
The leaf necrosis
appeared to be most prevalent under conditions of low temperature and
high humidity.
Experiments were made to determine the relationship of
the leaf necrosis to the four strains of SCMV.
Ten beefbuilder sorghum
seedlings were inoculated with strains A, B-,-D, and H.
The inoculum
used was standardized by increasing in sorghum for three successive
generations.
After inoculation,
the plants were placed in a Sherrer-
Gillete controlled environment chamber for symptom development.
The
environment of the chamber consisted of an air temperature of 85°F,
2000 ft. candles of light.
V..
A.
--
Physical Properties of Strains of SCMV
Thermal inactivation point (TIP)
Infected tissue of each strain which had been increased in sorghum
for 15 days was harvested and ground in a fruit grinder.
One ml ali­
quots of the undiluted juice were placed in 5 ml serological tubes
which had been preheated.
Sterile 1 ml syringes were used to place
the material into the tubes with care taken to avoid splashing material
onto the sides of the tubes.
The tubes were immersed in a continuously
16
agitated water bath for 10 min at 49, 51, 53, 55, and 57°C, respec­
tively.
The tubes were then.cooled, and each sample was assayed on
40 sorghum seedlings.
B.
Dilution end point (DEP)
Dilution end points were determined for each strain of SCMV.
The inoculum used in these tests was standardized by increasing the
sorghum for three successive generations.
Infected tissue of each
strain was harvested for 15 days, weighed into 2 gm samples, and ground
in a mortar.
10"2, 10"3,
Aliquots of freshly expressed juice were diluted 10“*-,
and 10"^.
Each dilution was assayed on 100 sorghum
seedlings.
VI.
Comparison of Three Methods of Virus Purification
Three methods of purification were compared to determine which
yielded the greatest amount of infectious virus.
increased in sorghum for 3-4 weeks.
divided into 3 aliquots.
technique.
,
The tissue was chopped and
Each aliquot was purified by a separate
Assays were made to compare infectivity of the virus at
various stages of each procedure.
1)
Strain H was
.
The procedures used were as follows;
Pirone and Anzalone (57) described a method for purifying
SCMV from infected sorghum tissue (acid clarification method).. .One
hundred and fifty gm of sorghum tissue, collected 3 weeks after
inoculation, was blended in a Waring blendor with an equal volume
of .02 M sodium sulfite.
The supernatant was acidified with 1.0N HC1
to pH 4.7 and centrifuged in a Sorvall RC-2 centrifuge for 5 m i n at
5000 rpm.
The supernatant was then centrifuged at 27,000 rpra for
5 min using a number 30 rotor in a Spinco L-2 centrifuge.
The
resulting supernatant was centrifuged at 30,000 rpm for 1.5 hr.
The pellets were pooled and resuspended in 2 ml of .02M sodium sulfite
for 2-3 hr, using a magnetic stirrer.
The preparation was then cen ­
trifuged for 5 min at 5000 rpm, and the supernatant was centrifuged
for 1 min at 4000 r p m in a number 50 Spinco rotor using 3-ml tubes
and adapters.
The supernatant was layered onto a density-gradient
column p r e p a r e d ‘in 1.25 x 3.5 inch tubes by layering 10, 14, 14, and
14 ml of 10, 20, 30, and 40% sucrose, respectively,
in 0.02M sodium
sulfite, and centrifuged in an SW 25.2 rotor at 24,000 rpm for 1.5 hr.
The virus band was removed with an ISCO density-gradient fractionator.
Infectivity assays at each step in the purification procedure
were made by inoculating 50 sorghum seedlings with undiluted and with
10~1 and 10"2 dilutions of the virus.
2)
Shepherd (66) described a method for the purification o f a
mosaic virus of corn in California
cation method).
(chloroform-strong buffer clarifi­
One hundred and fifty gm of sorghum tissue, collected
3 weeks after inoculation, was blended in a Waring blender with an
equal volume of 0.5M sodium citrate containing 0.5% mercaptoethanol.
The juice was expressed through two thicknesses of cheesecloth.
An
equal volume of chloroform was added to the extract and the mixture
was shaken,
then centrifuged (10,000 rpm for 10 min) to recover the
aqueous phase.
This was followed by high speed centrifugation (30,000
rpm for 1.5 hr in a number 30 rotor of a Spinco L-2 centrifuge) of the
clarified extract.
Following high speed centrifugation, pellets were
resuspended as before,
except 0.005M borate, pH 8.2'was used as the
18
resuspending liquid.
at 5,000 rpm.
The preparation was then centrifuged for 5 min
The supernatant was given a high speed centrifugation
(1 min at 40,000 rpm) in a number 50 Spinco rotor using 3-ml tubes
and adapters.
The supernatant was layered onto sucrose density-
gradients as before, except,
borate, pH 8.7.
1.5 hr.
the gradients were made in 0.005M
The gradients were centrifuged at 25,000 rpm for
The virus band was removed using an ISC0 density-gradient
fractionator.
Infectivity assays were made at each step in the puri­
fication procedure.
3)
Delgado-Sanchez and Grogan (26) described a method for the
purification of potato virus Y from tobacco tissue (chloroform-water
clarification m et h o d ) .
This method was chosen for use with SCMV
because of the similarity in morphology and physical properties of
SCMV with other members of the potato virus Y group in Brandes1 (18)
system of classification.
The method was modified for use with SCMV. '
One hundred fifty gm of infected sorghum tissue collected 3 weeks
after inoculation, was blended with an equal volume of distilled
water containing 0.3% ascorbic acid,
sodium diethyldithiocarbamate (SDDC).
two thicknesses of cheesecloth.
.3% 2-mercaptoethanol and 0.01M
The juice was expressed through
An equal volume of chloroform was
added to the extract and the mixture was shaken, then centrifuged
(10,000 rpm for 10 min) to recover the aqueous phase.
This was fol­
lowed by high speed centrifugation (30,000 rpm for 1.5 hr in a
number 30 Spinco rotor of a Spinco L-2 centrifuge) of the clarified
extract.
After high speed centrifugation,
the pellets were resus­
pended in 0.1M borate, pH 8.2 containing 0.01M ethylenediamine
19
tetra-acetic acid (EDTA).
for 5 min at 5,000 rpm.
The resuspended material was centrifuged
The supernatant was given a high speed cen­
trifugation (1 m i n at 40,000 rpm) in a number 50 Spinco rotor using
3-ml tubes and adapters.
The supernatant was layered onto sucrose
density-gradients as before, except, the gradients were made in 0.005M
borate, p H 8.2.
The gradients containing the virus were centrifuged
for 1.5 hr at 25,000 rpm.. The virus band was removed with an ISCO
density-gradient fractionator.
Infectivity assays were made at each
step in the purification procedure.
VII.
A'.
Serology of Strains of SCMV
Production of antisera
Antiserum to SCMV was prepared by intravenous and intramuscular
injections
(10) of partially purified virus into a rabbit.
Virus
used as antigen was obtained using the modification of Delgado-Sanchez
and Grogan’s procedure for potato virus Y.
The virus was obtained
directly from the density-gradient column.
One ml of each of the four
SCMV strains and the Johnson grass strain of SCMV was injected intra­
venously into rabbits at the first injection.
Intramuscular injec­
tions of 0.5 ml of antigen and 0.5 ml of Freund's incomplete adjuvant
were also administered the first week, and continued at weekly inter­
vals for 5-6 weeks.
Serum was obtained 7 days after the last injection.
Dilution titration was used to determine the antiserum titers.
B.
Microprecipitin tests
Microprecipitin reactions under mineral oil were used as one of
the serological tests on the various sera and virus strains.
All
20
antisera and virus dilutions were made in 0.857® saline.
In these
tests, antiserum end points were determined by mixing partially puri­
fied virus with an equal volume of antisera diluted in twofold series.
The antigen-serum mixture was incubated overnight before observation
of the final results.
C.
Agar diffusion tests
SCMV, a long flexuous rod-shaped plant virus, will not diffuse
in agar gels.
However, Purcifull and Shepherd (61) devised a method
for degrading flexuous rod-shaped virus particles into small antigenically active fragments which diffuse readily in agar gels.
The
method used in these studies was a modification of Purcifull and
Shepherd's.
About 150 gm of infected tissue of each strain of SCMV,
including the Johnson grass strain of SCMV, was partially purified
using the modification of Delgado-Sanchez and Grogan's method for
potato virus Y.
After clarification and high speed centrifugation,
the pellets were resuspended in 2 ml of distilled water.
tion was then centrifuged for 5 min at 5,000 rpm.
The prepara­
The clarified
supernatant of each strain was added to an equal volume of .'1M
ethanolamine, pH 10.5, in order to degrade the'virus particles into
antigenically active fragments
(60).
Agar gel double diffusion tests were done in plastic petri dishes
(Falcon Plastics, Division of B-D Laboratories, Los Angeles, Calif.).
Eleven ml of 0.85% Ion Agar No. 2 (Consolidated Laboratories, Chicago
Heights,
111.) containing 0.85% sodium chloride and 0.4% sodium azide
were poured into each standard 9 cm dish.
Two patterns were used in
21
these tests.
One pattern had a center well 7 ntm in diameter sur­
rounded by 8 peripheral wells of 4 mm. diameter at a distance of 7 m m
from the edge of the central one.
All wells held approximately 0.2 ml.
Antiserura was placed in the central well and the peripheral wells
were filled with antigen.
Another pattern consisted of a center well
7 m m in diameter surrounded by four equally spaced peripheral wells of
4 m m diameter at a distance of 7 m m from the edge of the central one.
Antigen was placed in the central well and
with the antisera.
the outer wells were filled
During formation of precipitin zones, the dif f u ­
sion plates were kept at room temperature in a moist chamber.
Zones
were most easily observed with horizontal illumination against a
black background.
VIII.
Soil Transmission Studies
In the course of bioassays on strain H of SCMV, noninoculated
sorghum seedlings often became infected when grown in greenhouse flats
which contained infected seedlings.
The frequent occurrence of the
phenomenon under screened greenhouse conditions,
aphids,
in the absence o f
suggested that some method of spread other than by aphids
might be responsible.
A.
Seed transmission studies
Sorghum grains were germinated in greenhouse flats and allowed
to grow for 1-2 months.
mosaic symptoms.
Seedlings were examined periodically for
22
B.
Soil transmission studies
In initial tests,
greenhouse flats.
sorghum grains were planted in rows in
At the two-leaf stage, alternate rows were inocu­
lated with strain H of SCMV.
Controls were sorghum plants grown in
porcelain pans to keep their root systems separate from those of
inoculated plants.
The pans containing the control plants were placed
in the center of a flat containing inoculated plants.
This allowed
the control plants to be near the inoculated plants in order to check
for possible aphid transmission.
Preliminary tests indicated that root contact was not neces­
sary in order to obtain transmission.
Two types of tests were, ,s.gt......
up to test whether root contact was necessary for transmission.
The
first method, which will be referred to as Method A, consisted of
placing porcelain pans containing noninoculated seedlings in flats as
described previously.
The pans were set below the soil level to allow
water to run from the flat containing the infected plants into the pan
containing the noninoculated plants.
The controls were set up in the
same manner except the porcelain pans were set well above the soil
level.
Care was taken to avoid water splashing into the controls
when the infected plants were watered.
Another test (Method B) involved planting sorghum grains in
4-inch peat pots at either end of an 8” x 4" plastic container.
Plants at one end of t.he container were inoculated with SCMV at the
two-leaf stage, while the plants at the other end were left non­
inoculated.
Since there was no soil between the peat pots, roots
could be kept separate and under observation.
The only contact
between the plants was by means of water at the bottom of the con­
tainer.
Experiments mentioned previously (Method A and Method B) were
also made under screened cages.
The tests were set up as before under
32 mesh screen cages in a screened greenhouse.
Cages were sprayed
with phosdrin and plants were germinated under the cages and inocu­
lated as in other tests.
•
EXPERIMENTAL RESULTS
I.
Reaction of Virus Strains on Standard Differential Varieties
Table 1 shows the infection ratio when the standard differentials
were inoculated with each virus strain.
high infection ratio.
All strains of SCMV showed a
The differential varieties inoculated with the.
Johnson grass mosaic did not become infected.
Strain B could be differentiated from the other strains quitereadily.
Symptoms produced were typical of those described by Abbott
and Tippett
(5).
The symptoms produced by strain D were also differ­
ent from those produced by strains A, B and H.
However, they were not
typical of those described by Abbott arid Tippett.
Strains A and H
produced similar symptoms on the differential varieties and could not
be differentiated.
II.
Symptoms on Sorghum Produced by the Different Strains
The leaf necrosis developed on plants infected with strains A,
D, and H, but not with strain B (Plate 1).
Necrosis was most severe
on plants infected with strains A and H.
III.
A.
Physical Properties of Strains of SCMV.
Thermal inactivation points
Thermal inactivation studies were made to determine if a differ­
ence existed among strains of SCMV in their tolerance to heat.
data in Table 2 show the results of three experiments.
SCMV were active at 55°C, but inactive at 57°C.
24
•
The
All strains of
25
Table 1.
Results of inoculation experiments showing infection ratio
of strains A, B, D, H, and Johnson grass mosaic in standard
differentials.
Strain
Differential variety
A
B
D
H
Jb
P.O.J. 234
7/73
7/7
4/7
7/7
0/5
C.P. 31-294
7/7
5/7
2/7
7/7
0/6
C.P. 31-588
7/7
6/7
6/7
6/6
0/6
g
Denominator, number of plants inoculated; numerator, number of
plants infected.
^Johnson grass mosaic.
Plate 1.
Reaction of beefbuilder sorghum to strains A, B, D
and H of sugarcane mosaic.virus (SCMV) when grown
under conditions of low temperature and high
humidity.
Left to right strains A, B, D, and H.
26
27
Table 2.
Thermal inactivation points of strains A, B, D, and H
of SCMV in sorghum.3
Temperature °C.
Virus strain
49°
51°
Strain A
78b
Strain B
53°
55°
57°
73
5
1
0
63
38
13
5
0
Strain D
98
53
12
1
0.
Strain H
98
35
18
9
0
3
Results of three experiments.
bData expressed as per cent of plants infected.
28
B.
Dilution end points
Dilution end point studies were made to determine if differ­
ences existed among strains in their tolerance to dilution.
results of three experiments are shown in Table 3.
The
The data indi­
cate a difference among the strains in their tolerance to dilution.
Strains A and H were still infectious at 1 0 ~ \
strain B at 10
-2
strain D at 10"^, and
.
IV.
Comparison of Three Methods of Virus Purification
Preliminary attempts to purify the four strains by the acidi­
fication method indicated that strain B could not be purified by
this technique.
A comparative study was made of three purification
methods to determine which procedure would give the highest yield of
infectious virus,
and which could be used for all strains.
Assays
were made of aliquots taken at different steps in each procedure.
The steps which were assayed are shown in Table 4.
Table 5 shows
the results of the three methods of purification.
With each procedure,
the virus banded in the gradients in a
distinct zone about 22 m m from the miniscus.
As a measure of purity,
ultraviolet absorption spectra of the material taken from the gradients
were determined.
A typical ultraviolet absorption spectrum was not
obtained with material resulting from the
nor from the
acid clarification method,
chloroform-buffer clarification method.
However,
an
absorption spectrum typical for nucleoproteins, with the absorption
maxima and minima near 260 and 240 mu, E ^ 2 . = 1.24 and E
= 1.06,
280
min
*
was obtained with material purified with the
fication method
(Figure 1).
chlqroform-water clari­
29
Table 3.
Dilution end point of strains A, B, D, and H of SCMV in
sorghum.3
Dilution
r— 1
39b
14
8
1
25
<1
0
0
Strain D
50
7
3
0
Strain H
.33
8
4
1
1— 4
!
O
10"2
Virus strain
Strain A
Strain B
10"3
io-4
aResults of three experiments.
^Data expressed as per cent of plants infected at each dilution.
Table 4.
Procedures used in purification by different methods after
which assays were made.
Procedure
Treatment
1)
Homogenization
Acid
clarification
.02M N a 2S03
Chloroformbuffer
Chloroform' water
0.5M Na
citrate, 0.5%
mercaptoethanol
1
2)
Clarification
3)
Resuspended
pellet after 1.5
hr
4)
L ow speed
5)
Density gradient
centrifugation
pH adjusted to
4.7, low speed
(5,000 rpm 5 min)
high speed (5 min
27,000 rpm)
Resuspended in
•02M N a 2S03 '
5 m i n at 5,000 rpm,
1 min at 40,000 rpm
1.5 hr at 25,000
rpm
Chloroform,
low speed (10 mi n
10,000 rpm)
Resuspended in
0.005M borate, pH 8.2
0.3% Ascorbic
acid, 0.01M sodium
diethyldithio
carbamate, 0.3%
2-mercaptoethanol
Chloroform,
low speed (10 min,
10,000 rpm)
Resuspended in
0.1M borate
0.01M EDTA, pH 8.2
same
same
same
same
31
Table 5. ■ Comparative infectivity p f SCMV after treatments listed in
Table ’3.a
•
Treatments
1)
Homogenization
2)
Acid
clarification
Procedure
Chloroformbuffer
Chloroformwater
42
19
•38
Clarification
8
3
33
3)
Resuspension
2
4
20
4)
Low speed
10
3
10
5)
Density gradient
2
2
.
.
15
aAverage of two experiments.
Data expressed as per cent o f plants
infected at a dilution of lO"*-.
32
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
220
240
260
280
Wavelength (Mu)
Figure 1.
Absorption spectrum of a purified preparation of sugarcane
mosaic virus.
The curve is a tracing using a Perkin Elmer
Spectrophotometer.
33
V.
Usefulness of the Three Procedures for Purification
of Different Strains
Using the scanning patterns from the density-gradient fractionator as a measure of the relative virus yields, a difference was noted
with several of the strains in their reaction to the three methods of
purification.
Strains A, D and H gave relatively high yields with
each purification procedure.
Numerous attempts to purify strain B and
the Johnson grass strain with the
acid clarification method
failed,.
However, both strain B and the Johnson grass strain gave relatively
high yields of virus when purified with either the
buffer clarification method,
or the
chloroform-strong
chloroform-water clarification
method.
VI.
A.
Serology of Strains of SCMV
Microprecipitin tests
Antisera to strains A, B, D, and H were prepared and tested with
the respective homologous and heterologous antigens.
In addition,
the
Johnson grass strain of SCMV present in Louisiana was tested against
antiserum to strain H to determine its relationship to SCMV.
Healthy
sorghum protein tested against all antisera served as the control.
The precipitates obtained with the antisera and the partially purified
viruses were of the open flocculent type characteristic of rod-shaped
plant viruses.
The precipitin end points with antisera to strains A, B, D, and
H were 1/128,
1/256,
1/128, and 1/256, respectively, when antisera
reacted with the homologous antigens (Table 6).
The heterologous
precipitin end point showed slight variations, but never varied more
Table 6.
Microprecipitin reactions of sugarcane mosaic virus
strains A, B, D, and H.
Antigen
St. A-SCMV
ii
ii
ii
St. B-SCMV
ii
it
ii
St. D-SCMV
ii
ii
ii
St. H-SCMV
ii
ii
ii
JGM-St.SCMV
Antiserum
to:
8
++
Relative amounts of precipitate with various serum dilutions .
(as reciprocals)3 .
•
Normal
Antiserum
serum
32
512 16
64
128
0
4
256
-H++
St.
St.
St.
St.
A-SCMV
B- 11
D- 11
H- "
+ +
+ +
-H-
++
St.
St.
St.
St.
B-SCMV
A- "
D- «
H-"
++
++
++
-H-
++
++
++
St.
St.
St.
St.
D-SCMV
A- 11
B- 11
H- "
St.
St.
St.
St.
H-SCMV
A- "
B- "
D- "
St. H-SCMVb
Healthy sor­
ghum protein St.
ii
St.
ii
St.
ii
St.
A-SCMV
B- "
D- "
H- 11
+ +
+ +
+ +
+
+ +
■Hr
+ +
++
•H-H-'
+ +
+ +
-H++
-H-
-H-
-H-
-H-
+ +
+ +
+ +
+ +
++
-H- •
++•
+ +
+ +
+
++
++
++
+
-H-
+ +
(SCMV) strains against antisera to
'
+
+
+
+
mm
mm
mm
+
-
-
+
mm
-
-
-
+
+
-
mm
mm
4+
+
+
+
+
+
+
+
+
mm
m
-
-
-
-
-
mm
-
-
+
mm
-
mm
+
+
_
mm
+
+
mm
mm
■I
-
-H-
+
+
-
mm
•A
+ +
+
+
-
-
-
+ +
+
mm
-
mm
mm
mm
-
■a
mm
aa
+ +
+
-
+ +
-H-
+
+
+ +
+ +
+ +
+
+
-
-
-
-
mm
-
-
-
mm
mm
+
+
+
+
-
mm
-
mm
M
M
-
-
-
-
-
+
-
mm
-
-
••
mm
-
mm
+
-
-
mm
“
-
-
-
-
a+ + = Maximum precipitate; - = no precipitate.
cD a t a shown is an average of three experiments.
■
-
bJohnson grass strain of SCMV.
mm
35
than one dilution from the homologous reaction, except the heterolo­
gous reaction involving strain H antigen and strain A antiserum.
The
Johnson grass strain gave a precipitin end point of 1/64 when reacted
against strain H antiserum.
The antisera reacted with healthy sorghum
protein up to 1/8.
this precipitate was easily distinguished
However,
from that produced by the virus.
There was no reaction of the viruses
against normal serum.
These results show that strains A, B, D, and H are closely related
serologically and cannot be differentiated in microprecipitin tests.
These results also show that the Johnson grass strain of SCMV in
Louisiana is serologically related to SCMV, but not as closely as the
strains are to each other.
B.
Agar diffusion tests
Figures 2-10 show the results of agar.diffusion tests.
When the
antisera of strains A, B, D, and 11 were tested against respective
homologous and heterologous antigens, all antisera reacted against
antigens of strains A, D and H (Figures 2-10).
The zones produced
by these antigens banded together indicating that they were closely
related serologically.
Antigen of strain B did not react against any
of the antisera, including its own, indicating that it was probably
present in too low a concentration to detect.
Antigen of Johnson
grass mosaic gave a very weak reaction against antisera to strains of
SCMV and spurring occurred (Figure 10).
Figures 2-5.
Agar diffusion tests with strains of SCMV.
2) Precipitin bands produced between peripheral
wells containing degraded virus protein of strains
A, B, D, H and Johnson grass mosaic and healthy
sorghum protein and the center well containing
antiserum against strain A.
3) Center well con­
taining antiserum against strain B.
4) Center well
containing antiserum to strain D.
5) Center well
containing antiserum against strain H.
36
Figures 6-10. Agar diffusion tests with strains of SCMV.
6) Pre­
cipitin bands produced between peripheral wells
containing antisera against strains A, B, D, and H
and Johnson grass mosaic and the center well contain­
ing degraded virus protein of strain A.
7) Center
well containing degraded virus protein of strain B.
8) Center well containing degraded virus protein of
strain D.
9) Center well containing degraded virus
protein of strain H.
10) Center well containing
degraded virus protein of strain H.
37
•A
H
B
A
H
B
.
B
D
D
7
6
h
:d :
:A
w
H
a«-
B
8
9
H
d
10
:H:
b
B
38
VII.
A.
Soil Transmission Studies
Seed transmission
Preliminary tests indicated that SCMV was being transmitted
from infected plants to noninoculated plants in the absence of an
aerial vector.
Experiments were made to determine if the virus was
being transmitted through the seed.
A total of 1168 sorghum seedlings were examined for possible
seed transmission.
Of the seedlings tested, no infected plants were
obtained.
B.
Soil transmission
1.
Initial transmission tests
Seed transmission tests indicated that the virus was not being
transmitted through the seed.
Tests were then made to determine if
the virus was being transmitted through the soil in some manner.
Initial tests were designed to show that the virus was being transmitted through the soil.
Initial transmission tests were repeated
nine times.
The data in.Xab.le 7 sljow the results of these tests.
first five experiments,
transmission ranged from .7 to 5.4%.
In the
Nine
to twenty days were required for transmission, with nine days being
the shortest period of time in which transmission was obtained.
the first five experiments,
In
two plants became infected in the controls.
It seemed possible that this was due to contamination since no attempt
was made to avoid water splashing into the pans containing the con­
trol plants.
In the last four experiments, care was taken to avoid
39
Table 7.
Test
Transmission of sugarcane mosaic virus (SCMV) from infected
plants to adjacent noninoculated sorghum plants.
Test Plants
Infected
Exposed
%
Transmission
Check Plants
Exposed
Infected
1
201
11
5.4
138
0
2
288
8
2.7
141
1
3
318
13
4.1
60
0
273
10
3.7
138
1
5
286
2
.7
185
0
6
254
2
.8
193
0
7
288
9
3.1
141
0
8
200
10
5.0
154
0
9
399
. 18
4.5
702
0
. 4_ _
.
•
water splashing into the controls.
40
In these experiments,
mission to the test plants ranged from .8 to 5%.
trans­
There was no
transmission to the controls.
2.
Transmission in the absence of root contact
The fact that contamination occurred in the controls suggested
that root contact was possibly not necessary in order to obtain
transmission.
To determine if root contact was necessary,
two types
of tests (Method A and Method B) were made.
The data in Table 8 show the results of Method A.
Transmission
to plants grown in soil adjacent to inoculated plants was 4.5 to 8.8%.
Transmission to plants grown in porcelain pans into which water was
allowed to wash was 6.2%.
there was no transmission.
In pans which were watered separately,
Root contact was absent in both cases.
Data in Table 9 show the results of Method B. . Transmission was
about 5% in all replications.
There was no transmission to the con- '
trols.
3.
Transmission under screen cages
Tests using methods A and B were made under 32 mesh screen cages
in a screened greenhouse.
Data in Table 10 show that transmission
ranged from 1.6 to 10.1% with Method A.
Transmission ranged from .9
to 2.5% with Method B.
4.
Identity of the virus being transmitted.
Serological, and infectivity tests and E M preparations of leaf
dip preparations confirmed that the virus being transmitted through
the soil was SCMV.
41
Table 8.
Transmission of sugarcane mosaic virus (SCMV) from infected
plants to noninoculated sorghum plants through soil water
(Method A).
Transmission to
Plants Outside Pan
Plants Inside Pan
Soil Water Allowed
to Wash into Pan
22/248
(8.8%)
10/160
Soil Water Kept Out
of Pan
20/442
(4.5%)
0/203
(6.2%)
(
0%)
Filmed as received
without page(s)
UNIVERSITY MICROFILMS.
43
Table 10.
Transmission of sugarcane mosaic virus (SCMV) in sorghum
plants under screened cages in the absence of root contact.
Method of
avoiding
root contact
Plants
Rep
Exposed
Infected
%
Transmission
A
1
2
3
•4
59
72
68
66
6
6
3
1
10.1
8.3
4.4
1.6
B
1
2
226
232
2
6
.9
2.5
44
5.
Attempts to determine factors associated with transmission.
In attempts to correlate certain factors with transmission,
sorghum grains were planted in steamed soil which had been autoclaved
for 8 hr.
In some instances,
lized prior to planting.
necrosis developed.
the sorghum grains were surface steri­
In all tests, a severe root and lower stem
Similar symptoms were observed w hen surface
sterilized seed were germinated in sterile distilled water, and on
water agar and potato dextrose agar (Plate 2).
Of several varieties
of sorghum tested, all produced the severe root necrosis.
Transmis­
sion was generally correlated with the severity of the root necrosis.
Nematodes were detected in soil water extracts, however, none
of these were plant-parasitic species.
Attempts to isolate fungi or to detect them in the thin sections
were negative.
Bacteria were isolated in some instances, but it was
impossible to determine their pathogenicity since the necrosis
occurred spontaneously,
lized.
even in seed which had been surface steri­
Plate 2.
Necrosis on roots of surface sterilized sorghum seeds
germinated in sterile water.
DISCUSSION
Since 1926 when McKinney (51) first demonstrated the existence
of strains of tobacco mosaic virus (TMV), every virus that has been
studied in detail has been found to exist in a range of forms or
strains.
Virus strains have been identified using a wide range of
criteria such as reaction of differential host varieties, physical,
chemical,
and biological properties, particle length, and morphology.
Several strains of sugarcane mosaic virus have been described
(2, 74).
Summers
(72, 73), and Summers, Brandes and Rands
(74) dif­
ferentiated strains of SCMV on the basis of their reaction on certain
differential host varieties.
differentiation were:
The characters used as a basis for strain
(1) nature of chlorosis,
absence of necrosis in the lesions,
(4)
growth retardation,
(2) presence or
(3) leaf sheath discoloration,
(5) germination recovery,
tiousness, and (7) length of incubation period.
(6) relative infec­
Although a number of
strains were differentiated by these workers, only four strains (A, B,
D, H) have been identified in Louisiana since 1950 (5).
Recently,
Abbott and Tippett (5) reported that these strains could be differen­
tiated on the differential host varieties C.P. 31-294 and C.P. 31-588
using the type of mosaic pattern as the principal diagnostic character.
According to Price
(59), unless the environmental conditions are care­
fully controlled it is difficult to compare .symptoms.
47
In the experiments reported in this dissertation,
in which each
of the four strains was introduced into the standard differentials,
only strain B showed symptoms similar to those described by Abbott
and Tippett
(5).
Strain D could also be differentiated from the
other strains, but symptoms were not typical of those described by
Abbott and Tippett.
Strains A. and H showed similar symptoms on the
differential varieties and could not be differentiated.
Since there
has been no attempt to describe symptoms under carefully controlled
conditions,
symptom expression should be interpreted with caution.
I.
Physical Properties of Strains of SCMV
Thermal inactivation studies showed that all strains of SCMV
were infectious at 55°C, but not at 57°C.
Adsuar
These results agree with
(6), and Costa and Penteado (23) who reported the same TIP.
These results do not agree with those of Chona (22), Abbott (1), and
Abbott and Tippett (5).
respectively,
Chona reported TIP's of 45, 55, and 65°C,
for three collections of SCMV in India.
However, the
figure of 65°C is much higher than that reported by other investiga­
tors
(1, 6, 23).
Abbott (1) in 1953 reported that all strains of
o
SCMV were inactivated at 53 C.
(5)
In a recent study, Abbott and Tippett
reinvestigated the physical properties of three strains
and eight variants of these strains.
H were 53, 52, and 49°C, respectively.
(A, D, H)
The TIP's for strains A, D and
However,
the fact that inacti­
vation was obtained at one temperature and activity regained at a
higher temperature leads this author to question the validity of such
results.
•
48
Dilution end point studies indicate a difference among the four
strains of SCMV in their tolerance to dilution.
Strains A and H
retained infectivity at dilutions of 10“^, D at 10“^ and B at 10”^.
The relative concentration of strain B is 100 fold less than D.
Thus,
it appears that the DEP can be used to separate strain B from
the other three strains.
The severity of leaf necrosis in plants infected with certain of
the strains might be associated with virus concentration.
The lower
relative concentration of strain B could explain the absence of leaf
necrosis in plants infected with this strain.
The high relative con­
centration of strain A could explain the severity of the leaf necrosis
on plants infected with this strain.
It is also possible that produc­
tion of necrosis is a property of the virus-host interaction not
dependent on concentration.
In any event, it may be possible to use
the degree of leaf necrosis on beefbuilder sorghum to distinguish
strain B from other strains..
Table 5 shows the results of a comparative study of three methods
of purification for SCMV.
The data show that the chloroform-water
procedure gave the highest amount of infectious virus.
With the acid .
clarification and the chloroform buffer procedures, a major loss of
infectivity occurred.
The loss of infectivity can probably be attri­
buted to virus aggregation.
et al.
Shepherd and Pound (67), van Regenmortel
(81) and Delgado-Sanchez and Grogan (26) found aggregation a major
problem during extraction and purification of viruses in the potato
virus Y (PVY) group.
Van Regenmortel (80), reported that considerable
packing occurs with the rod-shaped.plant viruses during centrifugation.
•
According to Delgado-Sanchez and Grogan (26), their procedure reduced
the amount of aggregation of PVY considerably.
This may explain the
higher amount of infectivity obtained with SCMV using this method.
It is interesting to note that with the chloroform-buffer pro­
cedure infectivity was lower in the initial step (homogenization)
than with the other two procedures.
With this procedure,
was homogenized in 0.5M sodium citrate.
the tissue
According to Scott (64), some
viruses will break up into subunits if the ionic strength of the buffer
.-t
used approaches or exceeds 0.2M.
This may explain the lower infectivity
in the initial step with this procedure.
It is also interesting to note that chloroform clarification
appeared to reduce infectivity with the chloroform-buffer procedure,
but not with the chloroform-water procedure.
This difference may be
due to the presence of ascorbic acid and SDDC in the latter procedure.
Apparently these materials protect the virus particles in some manner
from chloroform inactivation.
Typical ultraviolet absorption spectrums were not obtained with
material resulting from either the acid or the chloroform-buffer pro­
cedure.
However,
typical U V absorption spectrums were obtained with
material resulting from the chloroform-water procedure.
This indicates
that virus obtained using the latter procedure had less host con­
taminating material than with the other two procedures.
Differences were observed between the various strains and their
reaction to the different purification procedures.
Using scanning
patterns from the density-gradient fractionator as a measure of rela­
tive virus yields,
Strains A, D, and H gave relatively high yields
50
with each purification procedure.
Numerous attempts at purification
of strain B and the Johnson grass mosaic with the acid- clarification
method failed.
The failure to purify strain B with this method could
also be due to the low concentration of strain B in the host plant.
Since greater losses are incurred with the acid procedure and the
chloroform-buffer procedure, virus present in low concentration ini­
tially might all be lost during purification.
This could also account
for the failure to purify the Johnson grass mosaic with this procedure.
Another possibility is that strain B and the Johnson grass mosaic may
have properties'different from those of the other three strains.
More
aggregation may have occurred using this method of purification.
II.
Serology of Strains of SCMV
Data in Table 6 show the results of microprecipitin tests of
strains A, B, D, and H of SCMV.
The Johnson grass strain was included
to determine its relationship to SCMV.
The homologous titers of the
antisera to strains A, B, D, and H were 1/128, 1/256,
respectively.
1/125, and 1/256,
The heterologous precipitin end point showed slight
variations, but never varied more than one dilution from the homologous
reaction,
except the heterologous reaction involving strain H antigen
and strain A antiserum.
The antisera reacted with healthy sorghum
protein up to 1/8, but the precipitate was easily distinguished from
that produced by the virus.
These data indicate that strains A, B,
D and H are closely related serologically and cannot be differentiated
in microprecipitin tests.
The Johnson grass strain reacted with strain H antiserum up to
1/64 dilution.
Thus,
the Johnson grass mosaic present in Louisiana
51
is serologically related to SCMV, but not as closely as the strains
are to each other.
other workers
These results are similar to those reported by
(11, 83).
Figures 2-10 show the results of the agar gel diffusion tests.
The figures show the reaction of each antiserum with the respective
heterologous and homologous antigens.
When antisera of strains A,
B, D, and H were tested against the degraded virus (antigen) of the
four strains, all antisera reacted against antigens of strain A, D
and H. • The zones produced by these antigens banded together indicat­
ing complete serological identity.
Antigen of strain B did not react
to any antisera including its own, although its antiserum reacted
against A, D, and H antigens.
The low concentration of strain B in
the host plant as shown in DEP studies and the low concentration of
purified virus obtained could account for the failure to obtain a
reaction with this strain.
Again,
this may indicate a difference
between this strain and the other strains of SCMV.
Antigen of the
Johnson grass mosaic gave a very weak reaction with antisera to the
strains of SCMV and spurring occurred (Figure 10).
This indicates that
the Johnson grass mosaic has certain antigenic sites not common to the
other strains of SCMV.
Thus, it may be concluded that Johnson grass
mosaic in Louisiana is a serologically distinct strain of SCMV.
III.
Soil Transmission of SCMV
Data in Table 7 show the results of initial tests designed to
demonstrate soil transmission of SCMV.
transmission ranged from .7 to 5.4%.
In the first five experiments,
In the first five experiments,
two plants became infected in the controls.
It seems likely that this
was due to contamination, since no attempt was made to avoid water
splashing into the pans containing the control plants.
In the last
four experiments, care was taken to avoid water splashing into the
controls.
Here,
transmission to the test plants ranged from .8 to 5%.
There was no transmission to the control plants.
These data indicate
that transmission occurred from infected plants to noninoculated plaftts
through the soil.
In these experiments no attempt was made to keep
the roots of infected plants separate from those of the noninoculated
plants.
Thus, root contact could haye accounted for the transmission
in.these experiments.
According to Harrison (36), soil-borne viruses
.are those "with an underground method of natural spread which does not
depend simply on contact between tissues of infected and healthy
plants."
Data in Tables 8 and 9 show that root contact is not neces­
sary in order to obtain transmission.
In the first experiments
(Method
A), transmission to plants grown in soil adjacent to inoculated plants
was 4.5 and 8.8%.
Transmission of plants grown in porcelain pans into
which water was allowed to wash was 6.2%.
separately,
there was no transmission.
In pans which were watered
Data in Table 9 also demon­
strates that root contact is not necessary in order for transmission
to occur.
Transmission was about 5% in all replications.
Since SCMV is an aphid transmissible virus, experiments were made
under screen cages to rule out any possibility of aphid transmission.
Data in Table 10 show the results of this experiment.
transmission ranged from 1.6 to 10.1%.
ranged from .9 to 2.5%.
With Method A,
With Method B, transmission
53
The mechanism of transmission of SCMV through the soil is
unknown.
Attempts made to correlate the presence of a biological
vector with transmission were all negative.
A severe root and lower
stem necrosis was observed on most of the sorghum plants
(Plate 2).
There was a general correlation between the severity of the root
necrosis and transmission.
What role the root necrosis plays in
transmission is not known.
One possible explanation is that the virus
is simply released from infected plants into the soil.
This virus
could then enter noninoculated plants through the necrotic areas in
the roots.
Yarwood (87) reported virus release from roots of plants
infected with tobacco necrosis virus
(TMV).
(TNV) and tobacco mosaic virus
Grogan and Campbell (35) do not agree with the hypothesis that
soil transmission occurs through wounds produced by roots growing
through soil or sand.
These authors state that it is practically
impossible to maintain freedom from a fungus,
ordinary greenhouse conditions.
such as Olpidium, under .*
For this reason,
they are of the
opinion that accidental contamination could account for reported trans
mission of viruses in the absence of a biological vector.
SUMMARY
Studies were made to determine if strains A, B, D and H of SCMV
could be differentiated on the basis of physical, chemical and
biological properties.
Studies of the physical properties of the four strains showed that
thermal inactivation points
entiation.
(TIP) are of no value in strain differ­
All strains were still active at 55°C, but not at 57°C.
Dilution end point (DEP) studies showed a difference in certain of
the strains in their tolerance to dilution.
Strain A and H were
still infectious at 10"^, strain D at 10"^, strain B at 10~^.
A severe leaf necrosis developed on plants infected with certain
of the strains.
Necrosis occurred o n plants infected with strains
A, D, and H, but not on plants infected with strain B.
There..was
a correlation between the presence o f the leaf necrosis and virus
concentration.
Three methods of purification were compared to determine the one
best suited for use with SCMV.
A modification of the method of
Delgado- Sanchez and Grogan for potato virus Y (PVY) yielded the
highest amount of infectious virus.
Virus purified by this method
had less host contaminating material than with other methods tested
Microprecipitin tests could not be used to differentiate any of the
strains.
related.
In these tests, all strains appeared to be closely
55
7.
Agar diffusion tests showed that strains A, D, and H are closely
related.
Antigen of strain B did not react with antisera to any
of the strains including its own.
8.
Microprecipitin and agar diffusion tests showed that the Johnson
grass mosaic in Louisiana is serologically related to SCMV, but
not as closely as are the strains to each other.
Spurring in agar
diffusion tests showed that it is a distinct strain of SCMV.
9.
Studies showed that SCMV can be transmitted from infected plants
to noninoculated plants through the soil. Transmission occurred in
the absence of root contact.
The involvement of a biological
vector in soil transmission remains to be demonstrated.
LITERATURE CITED
1.
Abbott,
E. V. 1953. Tolerance to dilution and heat of six strains
of sugarcane mosaic.
Internatl. Soc. Sugar Cane Technol. Proc.
8: 911-913.
.2.
Abbott,
E. V. 1961... A new strain of
(Abstr.) Phytopathology 61: 642.
3.
Abbott,
E. V., and L. J. Charpentier.
vectors of sugarcane mosaic virus.
Technol. Proc. 11: 755-760.
4.
Abbott, E. V., and
R. L. Tippett.
1964. Additional hosts of
sugarcane mosaic virus.
Plant Disease Reptr. 48: 443.
5.
Abbott, E. V., and
R. L. Tippett.
1966. Strains of sugarcane
mosaic virus.
U.S.D.A. Tech. Bui. 1340, 25 p.
6.
Adsuar, J.
1950. On theproperties of sugarcane mosaic virus. .
Phytopathology 40: 214-216.
7.
Anzalone, L . , Jr. 1963.
Susceptibility of rice to a strain of
the sugarcane mosaic virus.
Plant Disease Reptr. 47: 583-584.
8.
Anzalone, L., Jr., and T. P. Pirone.
1964.
Transmission of
sugarcane mosaic by Myzus persicae. Plant Disease Reptr.
48: 984-985.
9.
Ball, E. M.
1961.
plant viruses.
10.
sugarcane mosaic virus.
1959.
Additional insect
Internatl. Soc. Sugar Cane
Serological tests for the identification of
Amer. Phytopathological Soc., Ithaca, N. Y.
Ball, E. M.
1964.
Serology:
Techniques used in plant virus
research, p. 235-252.
In M. K. Corbett and H. D. Sisler
(eds.)
Plant Virology.
Univ. Florida Press, Gainesville,
Florida.
11.
Bancroft, J. B., A. J. Ullstrup, M. Messieha, C. E. Bracker, and
T. E. Snazelle.
1966.
Some biological and p h ysical properties of a midwestern isolate of maize d w a r r ^ ^ ^ b g ^ v i r u s .
Phytopathology 56: 474-478.
12.
Bawden, F. C.
1964.
Plant viruses and virus diseases.
Ronald Press Co., N. Y.
361 p.
13.
Bennett, C. W.
1963.
Interactions between viruses and virus
strains.
Adv. in Virus Res. 1: 40-67.
56
The ':
57
14.
Bird, J., H. Cibes, and M. A. Tio.
1958.
Transmission of the
causal agent of chlorotic streak disease of sugarcane through
the roots of plants grown in nutrient solution.
Puerto Rico
Agr. Exp. Sta. Tech. Paper 27: 1-17.
15.
Birkeland, J. M.
1934.
Serological studies of plant viruses.
Bot. Gaz. 95: 419-436.-
16.
Brakke, M. K . , and A. P. Estes.
1966.
Correlation of Polymyxa
graroi’nis with transmission of soil-borne wheat mosaic virus.
Virology 28: 772-774.
17.
Brandes, E. W.
1920. Artificial and insect transmission of
sugarcane mosaic.
J. Agr. Res, 19: 131-138.
18.
Brandes, J.
1964.
Identifizierung von gestreckten pflanzenpathogenen Viren auf morphologischer Grundlage.
Mitt. Biol.
Bundensanstalt Land-Forstwirtsch.
Berlin-Dahlem 110: 5-130.
19.
Brandes, J., and C. Wetter.
1959.
Classification of elongated
plant viruses on the basis of particle morphology. .Virology
8: 99-115.
20.
Bruehl, G. W.,
and V. D. Damsteegt.
1961.
Soil-borne mosaic
of fall-seeded oats in western Washington.
Plant Disease
Reptr. 45: 884-888.
21.
Chester, K. S.
1936. Serological tests with Stanley's crystal­
line tobacco-mosaic protein.
Phytopathology 26: 715-734.
22.
23.
24.
Chona, B. L.. 1944.
Fmg. 4: 178-181.
Sugarcane mosaic and its control.
Costa, A. S., and M. P. Penteado.
1951.
plants for the sugarcane mosaic virus.
758-763.
Indian
Corn seedlings as test
Phytopathology 41:
Dale, J. L.
1964.
Isolation of a mechanically transmissible
virus from corn in Arkansas.
Plant Disease Reptr. 48: 661663.
25.
Dale, J. L., and L. Anzalone, Jr.
1965. Infection of sugarcane
with mechanically transmissible corn virus.
Plant Disease
Reptr. 49: 757-760.
26.
Delgado-Sanchez, S., and R. G. Grogan.
1966.
Purification and
properties of potato virus Y.. Phytopathology 56: 1397-1404.
27.
Desai, S. V.
1935.
The antigenic properties of the sugarcane
mosaic virus.
Current Sci. 3(7): 18. .
58
28.
Edgerton, C. W.
1955.
Sugarcane and its diseases.
State Univ. Press.
290 p.
29.
Freund, J.
1947.
Some aspects of active immunization.
In
C. E. Clifton (ed.), Vol. 1, Ann. Rev. of Microbiology,
p. 291-308.
Ann. Rev. Inc., Palo Alto, Calif.
30.
Fry, P. R.
1958.
Louisiana
The relationship of Olpidium brassicae (Wor.)
Dang, to the big-vein disease of lettuce.
Res. 1: 301-304.
New Zealand J. Agr.
31.
Fulton, R. W.
1967.
Purification and serology of rose mosaic
virus.
Phytopathology 57: 1197-1201.
32.
Gold, A. H., and J. P. Martin.
1955.
Electron microscopy of
particles associated with sugarcane mosaic.
Phytopathology.
45: 694.
33.
Grogan, R. C., F. W. Zink, W. B. Hewitt, and K. A. Kimble.
The association of Olpidium with the big-vein disease of
lettuce.
Phytopathology 48: 292-296.
34.
Grogan, R. G., and K. A. Kimble.
1964.
The relationship of
severe bean mosaic virus from Meico to southern bean mosaic
virus and its related strain in cowpea.
Phytopathology 54:
75-78.
35.
Grogan, R. G., and R. N. Campbell.
1966.
Fungi as vectors and
hosts of viruses.
In J. G. Horsfall (ed.), Vol. 4, Ann. Rev.
Phytopathology, p. 29-52, Ann. Rev., Inc., Palo Alto, Calif.
36.
Harrison, B. D.
1960.
The biology of soil-borne plant viruses.
Adv. in Virus Res. 7: 131-161.
37.
Harrison, B. D.
1964.
Transmission of plant viruses in soil,
p. 118-147.
In M. K. Corbett and H. D. Sisler (eds\) Plant
Virology. Univ. Florida Press, Gainesville, Florida.
38.
Herold, F., and J. Weibel.
1963.
Electron microscope demon­
stration o f sugarcane mosaic virus particles in cells of
Saccharum officinarum and Zea m a y s . Phytopathology 53:
469-471.
39.
Hewitt, W. B., J. D. Raski, and A. C. Goheen.
1958.
vector of soil-borne fanleaf virus of grapevines.
pathology 48: 586-595.
40.
Hidaki, Z.
1960.
The behavior of tobacco stunt virus in soils,
particularly supposing Olpidium brassicae (Wor.) Dang., as the
vector.
P r o c . Symp. Soil-borne Viruses. July. 1 960. (Scottish
Hort. Res. Inst., Dundee, Scotland).
1958.
Nematode
Phyto­
59
41.
Ingram,
J. W., and E. M. Summers.
1936.
Transmission of sugar­
cane mosaic by the rusty plum aphid, Hysteroneyra setariae.
J. Agr. Res. 52: 879-887.
42.
Ingram,
J. W . , and E. M. Summers.
1938.
Transmission of sugar­
cane mosaic by the green bug, Toxoptera graminum. J. Agr. Res.
56: 537-540.
43.
Jagger, I. C., and N. Chandler.
1934.
Big-vein,
lettuce.
Phytopathology 24: 1253-1256.
a disease of
44.
Janson,
B. F., L. E. Williams, W.R. Findley, E. J. Dollinger,
and C. W. Ellett.- 1965. Maize dwarf mosaic:
new corn virus
disease in Ohio.
Ohio Agr. Exp. Sta. Circ. 460.
16 p.
45.
Lawas, 0.M . , and W. L. Fernandez.
1949.
A
study of the trans­
mission of the corn mosaic and of the physical properties of
its virus.
Philippine Agr. 32: 231-328.
46.
Liu, S. P.
1950.
Studies of the sugar cane mosaic virus
in
Taiwan.
I.
Strains of the virus.
Taiwan Sugar Exp. Sta.
Rpt. 5: 72-98.
47.
Liu, S. P., and H. W. Li.
1953.
Studies on the sugar cane
mosaic virus in Taiwan. II.
The mode of resistance of cane
varieties and wild relatives to strains of mosaic.
Taiwan
Sugar Exp. Sta. Rpt. 10: 89-104.
48.
Lovisolo, 0., 0. Bodei and J. Volk.
1965.
Preliminary studies '
on the soil transmission of petunia asteroid mosaic virus
(= "Petunia" strain of tomato bushy stunt virus).
Phytopathol.
Z.
53: 323-342.
49.
Matz, J.
virus.
1933.
Artificial transmission of sugarcane mosaic
J. Agr. Res. 46: 821-839.
50.. McKinney, H.-H.
1923.
Investigations of the rosette disease
of wheat and its control.
J. Agr. Res. 23: 771-800.
51.
McKinney, H. H.
1926. Virus mixtures that may not be detected
in young tobacco plants.
Phytopathology 16: 893.
52.
McKinney, H. H.
1930. A mosaic of wheat transmissible to all
cereal species in the tribe Hordeae.
J. Agr. Res. 40: 547556.
53.
Miyamoto, Y.
1959.
The nature of soil transmission in. soilborne plant viruses. Virology 7: 250-251.
54.
Ouchterlony, 0.
1958. Diffusion-in-gel methods for immunological
analysis.
Progr. Allergy 5: 1-78.
60
55.
Perdomo, R., and I. L. Forbes.
1965.
St. Martinville grass(Raoul grass) susceptible to sugarcane mosaic virus.
Sugar.
• Bui. .44(2): 39.
56.
Perez, J., and J. Adsuar.
sugarcane mosaic virus.
9-15.
57.
Pirone, T. P., and L. Anzalone, Jr.
1966.
Purification and
electron microscopy of sugarcane mosaic virus.
Phytopathology
56: 371-372.
58.
Pirone, T. P., R. W. Toler, and W. P. Bond.
1967.
Mosaic
infected Johnson grass found in Louisiana.
Plant Disease
Reptr.
51: 108.
59.
Price, W. C.
1932. Acquired immunity to ring-spot in nicotiana.
Contrib. Boyce Thompson Inst. 4: 359.
1954.
Serological reactivity of
Puerto Rico Univ. Jour. Agr. 38:
60.
Purcifull, D. E., and J. F. Shepard.
1967.
Western celery mosaic
virus in Florida celery.
Plant Disease Reptr. 51: 502-505.
61.
Purcifull, D. E., and R. J. Shepherd.
1964.
Preparation of the
protein fragments of several rod-shaped plant viruses and their
use in agar-gel diffusion tests.
Phytopathology 54: 1102-1108.
62.
Purdy, H. A.
1928. Immunologic reactions with tobacco mosaic
virus.
Proc. Soc. Exptl. Biol. Med. 25: 702-703.
63.
Rafay, S. A.
1935. Physical properties of sugarcane mosaic
virus.
Indian J. Agr. Science 5: 663-670.
64.
Scott, H. A.
1963. Purification of cucumber mosaic virus.
Virology 20: 103-106.
65.
Scott, H.
A., M. Vincent, and W. J. Zaumeyer.
studies of red node and pod mottle viruses.
51: 755-758.
1961.
Serological
Phytopathology
66.
Shepherd, R. J.
1965.
Properties of a mosaic virus of corn and
Johnson grass and its relation to the sugarcane mosaic virus.
Phytopathology 55: 1250-1256.
67.
Shepherd, R. J., and G. S. Pound.
1960.
Purification, of turnip
mosaic virus.
Phytopathology 50: 797-803.
68.
Shepherd, R. J., and Q. L. Holdeman.
1965.
Seed transmission of
the Johnson grass strain of sugarcane mosaic virus in corn.
Plant Disease Reptr. 49: 468-469.
69.
Shepherd,
R. J., D. H. Hall, and D. H. Purcifull.
1964.
Occur­
rence of a new virus disease of corn in California.
Plant
Disease Reptr. 48: 749.
61
70.
Smith, K. M.
1937.
Further studies on a virus found in the
roots of certain normal-looking plants.
Parasitology 29:
86-95.
71.
Storey, H. H.
1927.
Strains of the viruses affecting the
graminae.
Internatl. Soc. Sugar Cane Technol. Proc. 2: 87-88.
72.
Summers, E. M.
1935.
Strains of the sugarcane mosaic virus in
Louisiana.
Internatl. Soc. Sugar Cane Technol. Proc. 5:
723-729.
73.
Summers, E. M.
1938. A study of common mosaic of sugarcane wijth
special reference to strains of the virus.
Internatl. Soc.
Sugar Cane Technol. Proc. 6: 564-565.
74.
Summers, E. M . , E. W. Brandes, and R. D. Rands.
1948.
Mosaic of
sugarcane in the United States with special reference to strains
of the virus.
U.S.D.A. Tech. Bui.
955 p.
75.
Tate, H. D., and S. R. Vanderiburg.
1939.
Transmission of
cane mosaic by aphids.
J. Agr. Res. 59: 73-79.
'76.
sugar­
Teakle, D. S.
1960.
Association of Olpidium brassicae and
tobacco necrosis virus.
Nature 188: 431-432.
77.
Tims. E. C., and C. W. Edgerton. 1931.
Behavior of
mosaic in
certain sugarcane varieties in Louisiana.
Amer. J. Bot. 18:
649-657.
78.
Tims, E. C., P. J. Mills, and C. W. Edgerton.
1935.
sugarcane mosaic in Louisiana. La. Agr. Exp. Sta.
39 p.
79.
. 80.
Studies on
Bui. 263,
Todd, E. H.
1964.
Sugarcane mosaic on St. Augustine grass in
Florida.
Plant Disease Reptr. 48: 442.
v an Regenmortel, M. H. V.
1964.
Separation of an antigenic plant
protein from preparations of plant viruses.
Phytopathology
54: 282-289.
81.
v a n Regenmortel, M. H. V., J. Brandes, and R. Bercks.
1962.
Investigations on-the properties of watermelon mosaic virus.
Phytopathol. Z. 45: 205-216.
82.
v a n Slogteren, D. H. M.
1955.
VIII. Serological microreactions
with plant viruses under paraffin oil, p. 51-54.
Proc. 2nd
Conf. Potato Virus Diseases, Lisse- Wageningen.
83.
Wagner, G. W., and J. L. Dale.
1966. A serological comparison
of maize dwarf mosaic virus isolates.
Phytopathology 56:
1422-1423.
62
84.
Williams, L. E., and L. J. Alexander.
virus isolated from corn in southern
Phytopathology 54: 912.
85.
Williams, L. E., and L. J. Alexander.
1965. Maize dwarf mosaic,
a new corn disease.
Phytopathology 55: 802-804.
86.
Willison, R. S., J. H. Tremaine, and M. Weintraub.
1961.
Sero­
logical and physical properties of some stone-fruit viruses:
non virus particles associated with infection.
Can. J. Bot.
39: 1447-1452.
87.
Yarwood, C. E.
1960. Release and preservation of virus by
roots.
Phytopathology 50: 111-114.
I
1964.
Ohio.
An unidentified
(Abstr.)
VITA
William Payton Bond was born November 18, 1941 in Franklinton,
La.
He received his elementary and secondary education at Franklinton
High School from which he was graduated in May, 1959.
Southeastern Louisiana College in September,
He entered
1959 and received his
B. S. degree in Dairy Manufacturing in May, 1963.
In September,
1963
he began study in the Department of Botany and Plant Pathology at
Louisiana State University.
He received his M. S. degree in Plant
Pathology in January,
He is presently a candidate for the
1966.
degree of Doctor of Philosophy in Plant Pathology.
63
EXAMINATION A N D THESIS REPORT
Candidate:
William Payton Bond
Major Field:
Plant Pathology
Title of Thesis:
Chemical,.Physical and Biological Properties of
Four Strains of Sugarcane Mosaic Virus
Approved:
Major Professor and Chairman
■tfOj-
■
Dean dr the Graduate School
EXAMINING COMMITTEE:
Im.'T
Date of Examination:
May 9, 1968